US5576181A - Process for selective removal of salivary α-amylase and assay for pancreatic α-amylase - Google Patents

Process for selective removal of salivary α-amylase and assay for pancreatic α-amylase Download PDF

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US5576181A
US5576181A US08/793,060 US79306091A US5576181A US 5576181 A US5576181 A US 5576181A US 79306091 A US79306091 A US 79306091A US 5576181 A US5576181 A US 5576181A
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amylase
salivary
pancreatic
antibody
beads
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David J. Torrens
Howard J. Marriage
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Euroapi UK Ltd
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Genzyme Ltd
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Assigned to GENZYME LTD. reassignment GENZYME LTD. CORRECTIVE ASSIGNMENT TO CORRECT THE SERIAL NUMBER IN ITEM 4, AS RECORDED ON REEL 6514 FRAME 832-835. Assignors: MARRIAGE, HOWARD J., TORRENS, DAVID J.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/823—Immunogenic carrier or carrier per se
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/824—Immunological separation techniques

Definitions

  • This invention relates to a process for the selective removal of salivary ⁇ -amylase and to an assay for pancreatic ⁇ -amylase.
  • the human pancreas contains a number of digestive enzymes, such as amylase, lipase and trypsin. Inflammation of this organ, such as in acute or chronic pancreatitis, may cause release of such enzymes into the bloodstream, where the increased activities thereof may be detected.
  • digestive enzymes such as amylase, lipase and trypsin.
  • Serum ⁇ -amylase is the most widely used clinical indicator of inflammation of or injury to the pancreas. It is of particular value in the differential diagnosis of acute abdominal pain of which acute pancreatitis may be the cause.
  • Amylase is usually determined by its ability to degrade a substrate generating a product which may be measured in a spectrophotometer. Such assays measure not only pancreatic-type amylase which is derived from the pancreas, but also salivary-type amylase, which is found in a number of tissues including salivary glands, testes, ovaries, fallopian tubes, striated muscle, lung and adipose tissue. Consequently, non-pancreatic disease may result in an elevation of total serum amylase activity.
  • Both the salivary and pancreatic enzymes are ⁇ -amylase (E.C.3.2.1.1.) which degrades 1,4-D-glucoside-linked oligo-and poly-saccharides by hydrolysis of 1,4- ⁇ -glucoside bonds to generate maltose and malto-oligosaccharides.
  • ⁇ -amylase E.C.3.2.1.1.
  • the primary structures of the two isozymes are very similar, they may be separated by electrophoresis, column chromatography, isoelectric focusing and radioimmunoassay, for example. However, these methods are slow and complex to perform.
  • Another known inhibition method uses two monoclonal antibodies "synergistically" to inhibit salivary amylase.
  • the main limitation of this procedure is that it is not possible to measure both total and pancreatic amylase using the same kit, as the vial containing the antibodies also contains ⁇ -glucosidase, which is necessary for the development of a coloured product. It has been suggested (Cummings and Fraser, Ann Clin Biochem, 26(4):335-340;1989), that the initial diagnosis of acute pancreatitis be established using pancreatic amylase, while the progress of the disease is followed by the measurement of total serum amylase.
  • the use of two different reagent systems would generally be regarded as wasteful of reagent and unnecessarily costly to the user.
  • a separation system requiring no involvement of chromatography or centrifugation which may be performed rapidly in an emergency assay may be envisaged where the antibody is immobilised to very large or magnetic particles.
  • the present invention concerns the use of monoclonal antibodies immobilised or coupled to physically-separable or separate supports to facilitate the removal of one antigen from a system so that another may be determined without significant interference.
  • the present invention provides a process for the selective removal of salivary ⁇ -amylase from a sample comprising salivary ⁇ -amylase and pancreatic ⁇ -amylase characterised in that there is used a monoclonal antibody against salivary ⁇ -amylase, which is immobilised or is coupled to a physically separable or separate support and which exhibits a binding affinity towards salivary ⁇ -amylase of at least 1 ⁇ 10 7 l/M and a cross-reactivity with pancreatic ⁇ -amylase of less than 1%.
  • Such physical removal of salivary ⁇ -amylase may, if desired, be followed by the determination of remaining pancreatic ⁇ -amylase, but may, of course, be an end in itself.
  • the monoclonal antibody exhibits a binding affinity towards salivary ⁇ -amylase of at least 1 ⁇ 10 8 l/M and a cross-reactivity with pancreatic ⁇ -amylase of less than 0.5%. More particularly, the monoclonal antibody is that identified as 5/262 (ECACC 90031302) or 5/330 (ECACC 90031306).
  • the monoclonal antibody is generally immobilised on a membrane or in a discrete layer or is coupled to a paddle, to a tube wall or to a particle, which may be separated by magnetism, centrifugation or filtration.
  • the present invention provides a kit for the selective removal of salivary ⁇ -amylase characterised in that it comprises such a monoclonal antibody.
  • a kit in accordance with the present invention may further comprises means for determining pancreatic ⁇ -amylase.
  • pancreatic ⁇ -amylase Of course, such are generally known.
  • the present invention provides a monoclonal antibody against salivary ⁇ -amylase characterised in that it exhibits the properties defined above and is preferably that identified as 5/262 (ECACC 90 031 302) or 5/330 (ECACC 90 031 306). Such may also be provided in accordance with the present invention in immobilised or coupled form as a reagent or even a "device”.
  • ECACC 90031302 and ECACC 90031306 have been deposited under the terms of the Budapest Treaty with the Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts. SP4 0JG, U.K.
  • An appropriate antibody against salivary amylase will bind rapidly and then remove it from solution, thus allowing, in this case, pancreatic amylase remaining in solution to be measured.
  • the key component of such a system would be regarded as the antibody, the development of a highly specific tight binding monoclonal antibody being preferred.
  • a procedure was undertaken to obtain monoclonal antibodies suitable for this technique using analytical procedures designed to generate and select for those having the tightest binding and lowest cross-reactivity. It is well known that the process for generating monoclonal antibodies of a particular type will result in many thousands of cell lines. Thus, the criteria for selection of suitable cell lines is an important part of the product development.
  • 5/262 and 5/330 two particularly tight binding monoclonal antibodies that were specific for salivary amylase were produced, referred to herein as 5/262 and 5/330, (90031302 and 90031306 respectively.) These had binding affinities towards salivary amylase of 3 ⁇ 10 8 l/M and 2 ⁇ 10 10 l/M, and cross-reactivities towards pancreatic amylase of less than 0.5% and less than 0.1%, respectively.
  • antibody 5/330 showed superior results for both binding affinity and specificity.
  • the two antibodies were coupled to a number of solid phase supports using different conjugation methods. Surprisingly, in each case where a direct comparison was made, the antibody with the weaker binding affinity in free solution showed superior ability to remove salivary amylase when bound to a solid phase. The cross-reactivities towards pancreatic amylase also behaved in an unexpected fashion. The antibody with the lowest cross-reactivity bound more pancreatic amylase when it was immobilised.
  • mice were immunised with immunopurified salivary amylase (purchased from Aalto Bioreagents, Dublin, Eire.) 50 ⁇ g of this material in Freund's complete adjuvant was injected via the intraperitoneal route into the mice. After three, six and nine weeks, the injections were repeated, but on these occasions Freund's incomplete adjuvant was used. On week eleven, 50 ⁇ g of the antigen in saline was injected intravenously. After a further three days, the animals were killed and the spleens removed.
  • immunopurified salivary amylase purchased from Aalto Bioreagents, Dublin, Eire.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • the myeloma cell line used was NSO (uncloned), obtained from the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK.
  • the myeloma cells, in logarithmic growth phase, were washed in DMEM and counted in a haemocytometer using phase contrast microscopy.
  • Spleen cells (1 ⁇ 10 8 ) were mixed with myeloma cells (7 ⁇ 10 7 ), centrifuged and the liquid removed. The resultant cell pellet was placed in a 37° C. water bath. Over a period of 1 minute, 1 ml of a 50% (w/v) solution of polyethylene glycol 1500 (PEG) in saline HEPES buffer, pH 7.5, was added and the mixture gently stirred for 11/2 minutes. Over a period of 5 minutes, 50 ml of serum-free DMEM was added, followed by centrifugation. The supernatant was discarded and the cell pellet re-suspended in 10 ml of DMEM containing 18% FCS.
  • PEG polyethylene glycol 1500
  • the resultant cell suspension was placed in each of 960 wells in an amount of 10 ⁇ l per well in standard multiwell tissue culture plates.
  • Each well contained 2 ml of standard HAT medium (hypoxanthine, aminopterin and thymidine), and a feeder layer of Balb/c macrophages at a concentration of 5 ⁇ 10 4 macrophages per well.
  • the wells were maintained at 37° C. under 9% CO 2 air at approximately 90% humidity.
  • Antibody binding was further assessed by radioimmunoassay.
  • Salivary amylase was labelled with 125 I by the chloramine T procedure to a specific activity of 70 ⁇ Ci/ ⁇ g.
  • antibody-bound tracer was separated from free tracer by donkey anti-mouse antibodies bound to a cellulose bead solid phase. From the radioimmunoassay, the percentage cross-reactivity with pancreatic amylase activity was obtained and, after Scatchard analysis, the binding affinities of the antibodies were determined.
  • tissue culture fluid was first passed through a 0.2 ⁇ m filter. To separate the antibodies, the fluid was pumped onto a column of immobilised protein A. After elution using a pH 3.5 glycine buffer, the antibody was dialysed into phosphate-buffered saline, pH 7.4.
  • Polystyrene beads having a diameter of 6.4 mm and coated with hydrazide groups were purchased from Life Science Laboratories (UK) Ltd.
  • a further gentle mixing of the beads was performed with 5.0 ml of 0.1M sodium bicarbonate containing approximately 1 mg of sodium borohydride. After a final washing with 100 ml of 0.1M sodium carbonate and 100 ml of deionised water, the beads were dried and stored at 4° C. prior to use.
  • the polystyrene beads coupled with antibody 5/262 were compared with those coupled with antibody 5/330 by incubating a single bead with 100 ⁇ l of either salivary or pancreatic amylase at an activity of 1000 u/l. The incubations were performed at room temperature for 1 hour. At the end of this period, 20 ⁇ l samples of the remaining liquid were assayed for amylase activity by a standard method.
  • the beads coupled with antibody 5/262 left 8% of the salivary amylase and 89% of the pancreatic amylase in solution. In comparison, 21% of the salivary amylase activity and 89% of the pancreatic amylase was left after incubation with the 5/330-coupled beads.
  • Dynabeads M-280 tosyl-activated are uniform superparamagnetic polystyrene beads having a diameter of 2.8 ⁇ m. The surface of these beads is pre-activated with a p-toluene-sulphonyl chloride treatment. This material was purchased from Dynal (UK) Ltd.
  • the pellet was re-suspended with 0.5 ml of 50 mM borate buffer, pH 9.5.
  • a solution of antibody at a concentration of 800 ⁇ g/ml was prepared in 50 mM borate buffer, pH 9.5, and then 500 ⁇ l of this was mixed with the re-suspended beads. This mixture was vigorously shaken at room temperature for 48 hours.
  • the antibody-coated beads were then washed three times, 10 minutes each time, with phosphate-buffered saline containing 0.1% bovine serum albumin. After another wash for 30 minutes, the beads were left to wash a last time overnight at 4° C., again in the same wash buffer. The coated beads were stored in the same buffer at 4° C. before being tested.
  • the beads coated with antibody 5/262 were compared with those coated with antibody 5/330 for their ability to remove salivary and pancreatic amylases from solution.
  • 20 ⁇ l of a 20 mg/ml suspension were mixed with 100 ⁇ l of salivary or pancreatic amylase at an activity of 1000 u/l.
  • the tubes containing the suspensions were placed over a magnet for 2 minutes. This drew the beads downwards to form a pellet, leaving a clear supernatant above.
  • 20 ⁇ l aliquots of the supernatant were taken for measurement of the residual amylase activity.
  • Dynabeads coupled with antibody 5/262 were found to have left 6% of the salivary amylase and 85% of the pancreatic amylase activity in the supernatant. However, the antibody 5/330-coated beads had left 12% of the salivary and 76% of the pancreatic enzyme in the unbound fraction.
  • Biomag magnetic beads are super-paramagnetic particles of iron oxide coated with polymeric silane to provide sterically unencombered functional groups. (The Biomag 4100 particles are obtainable from Advanced Magnetics, Cambridge, Mass., USA, and are more fully described in U.S. Pat. No. 4,554,088.)
  • the activated beads were washed with 4 ⁇ 500 ml 0.01M pyridine, pH 6.0.
  • 150 mg of antibody in 0.01M pyridine, pH 6.0 were mixed with the beads, resulting in a total volume of 150 ml. This was mixed overnight at room temperature. The particles were washed with 2 ⁇ 500 ml 0.01M pyridine, pH 6.0.
  • the particles were mixed with 700 ml of 1M ethanolamine, pH 8.0, for 3 hours at room temperature. To ensure complete removal of non-covalently bound antibody, the beads were subjected to a stringent washing protocol. The particles were first wahsed with 4 ⁇ 500 ml 50 mM glycine buffer, pH 10.0, with 1M NaCl and 0.01% w/v bronopol.
  • the particles were re-washed twice with the glycine NaCl buffer and then mixed with the same buffer for 3 hours. After four washes with 1M ethanolamine, pH 8.0, the particles were mixed overnight in the ethanolamine. The particles were then washed four times with phosphate-buffered saline containing 0.01% w/v bronopol and 0.1% w/v polypropylene glycol, and then re-suspended in the same buffer mixture to 5 mg/ml.
  • the beads were incorporated as part of the following reagent:
  • Bronopol was used to prevent bacterial growth, polypropylene glycol to prevent frothing during mixing with sample and urea to increase the recovery of pancreatic amylase by reducing non-specific binding.
  • Magnetic separation time was determined by the mixing of 100 ⁇ l particles suspension with 100 ⁇ l of 1000 u/l salivary amylase in phosphate-buffered saline containing 1% BSA in 1.5 ml tubes. After standing for 10 minutes at room temperature, the tubes were placed in a rack containing magnets composed of neodymium iron and boron for varying lengths of time. The removal of salivary amylase at greater than 98% occurred by 1 minutes as determined by analysis of the supernatant for amylase activity. This was performed using a benzylidine-blocked maltoheptaoside paranitrophenyl substrate using a kinetic microtitre plate analyser.
  • pancreatic amylase test solution 100 ⁇ l of 1000 u/l salivary and 100 ⁇ l of 1000 u/l pancreatic amylase test solution were mixed with 100 ⁇ l of reagent and allowed to stand for varying periods of time, followed by a 1 minute magnetic separation. The supernatant was analysed for the activity of pancreatic or salivary amylase. After 3 minutes, 97.6% of the salivary amylase was removed by the reagent, which increased to 98.2% with a 5 minute incubation. A further small increase in removal to 98.9% was observed after a 60 minute incubation. The recovery of pancreatic amylase was 88% at 3 minutes, 5 minutes and 60 minutes.
  • the capacity of the reagent system was tested by the incubation of 100 ⁇ l of various activities of either salivary or pancreatic amylase with 100 ⁇ l of reagent.
  • the incubation time was 5 minutes with a separation time of 1 minute.
  • the removal of salivary amylase was 98.0%, 97.2% and 96.7%, respectively, and the recovery of pancreatic amylase was 88%, 89% and 88%, respectively.

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US08/793,060 1990-11-16 1991-11-15 Process for selective removal of salivary α-amylase and assay for pancreatic α-amylase Expired - Fee Related US5576181A (en)

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EP (1) EP0486325B1 (da)
JP (1) JPH04267892A (da)
AT (1) ATE150178T1 (da)
AU (1) AU653182B2 (da)
CA (1) CA2055627A1 (da)
DE (1) DE69125100T2 (da)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092977A1 (en) * 2005-10-14 2007-04-26 Karl Reich Forensic test for human saliva

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EP2142663B1 (en) 2007-05-01 2016-08-10 Yissum Research Development Company of the Hebrew University of Jerusalem, Ltd. An assay and device for removing amylase from body fluids

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4554088A (en) * 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
US4939082A (en) * 1983-11-25 1990-07-03 Boehringer Mannheim Gmbh Process and monoclonal antibody for the specific determination of pancreas alpha-amylase in the presence of saliva alpha-amylase

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DE3500526A1 (de) * 1985-01-09 1986-07-10 Boehringer Mannheim Gmbh, 6800 Mannheim Spezifisch hemmender s-amylase-mak
DE3903114A1 (de) * 1989-02-02 1990-08-09 Boehringer Mannheim Gmbh Verfahren zur bestimmung eines enzyms aus einem isoenzymgemisch sowie hierfuer geeigneter testtraeger und dessen verwendung

Patent Citations (2)

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US4554088A (en) * 1983-05-12 1985-11-19 Advanced Magnetics Inc. Magnetic particles for use in separations
US4939082A (en) * 1983-11-25 1990-07-03 Boehringer Mannheim Gmbh Process and monoclonal antibody for the specific determination of pancreas alpha-amylase in the presence of saliva alpha-amylase

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
Bruns, D. E. et al, "Appendix: Production and Characterization of a Monoclonal Antibody to Distinguish Human Pancreatic and Salivary Amylase", Clin. Chem. 31(8) pp. 1286-1288, 1985.
Bruns, D. E. et al, Appendix: Production and Characterization of a Monoclonal Antibody to Distinguish Human Pancreatic and Salivary Amylase , Clin. Chem. 31(8) pp. 1286 1288, 1985. *
Gerber, M. et al, "A Monoclonal Antibody That Specifically Inhibits Human Salivary α-Amylase", Clin. Chem. 33(7) pp. 1158-1162, 1987.
Gerber, M. et al, A Monoclonal Antibody That Specifically Inhibits Human Salivary Amylase , Clin. Chem. 33(7) pp. 1158 1162, 1987. *
Hiroishi, S. et al, "Differnetial Assay of Salivary and Pancreatic α-Amylase in Serum and Urine, with Use of Monoclonal Antibody to Human Salivary Amylase Immobilized on Bacterial Cell Wall", Cli. Chem. 33(7) pp. 1235-1236, 1987.
Hiroishi, S. et al, Differnetial Assay of Salivary and Pancreatic Amylase in Serum and Urine, with Use of Monoclonal Antibody to Human Salivary Amylase Immobilized on Bacterial Cell Wall , Cli. Chem. 33(7) pp. 1235 1236, 1987. *
Ito, K. et al, "Preparation of Human Salivary α-Amylase Specific Monoclonal Antibody", J. Biochem. 97(5) pp. 1357-1362, 1985.
Ito, K. et al, Preparation of Human Salivary Amylase Specific Monoclonal Antibody , J. Biochem. 97(5) pp. 1357 1362, 1985. *
Mifflin, T. E. et al, "Rapid Quantitative, Specific Measurement of Pancreatic Amylase in Serum with Use of a Monoclonal Antibody", Clin. Chem. 31(8) pp. 1283-1288, 1985.
Mifflin, T. E. et al, Rapid Quantitative, Specific Measurement of Pancreatic Amylase in Serum with Use of a Monoclonal Antibody , Clin. Chem. 31(8) pp. 1283 1288, 1985. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092977A1 (en) * 2005-10-14 2007-04-26 Karl Reich Forensic test for human saliva

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EP0486325A3 (en) 1992-11-04
GB9024970D0 (en) 1991-01-02
DE69125100D1 (de) 1997-04-17
DK0486325T3 (da) 1997-04-01
JPH04267892A (ja) 1992-09-24
GR3023701T3 (en) 1997-09-30
EP0486325B1 (en) 1997-03-12
DE69125100T2 (de) 1997-06-19
EP0486325A2 (en) 1992-05-20
AU8781791A (en) 1993-04-08
CA2055627A1 (en) 1992-05-17
AU653182B2 (en) 1994-09-22
ATE150178T1 (de) 1997-03-15
ES2099139T3 (es) 1997-05-16

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